Glucosyl stevia composition

ABSTRACT

Glucosyl  stevia  compositions are prepared from steviol glycosides of  Stevia rebaudiana  Bertoni. The glucosylation was performed by cyclodextrin glucanotransferase using the starch as source of glucose residues. The compositions were purified to &gt;95% content of total steviol glycosides. The compositions can be used as sweetness enhancers, flavor enhancers and sweeteners in foods, beverages, cosmetics and pharmaceuticals.

FIELD OF THE INVENTION

The invention relates to a process for producing a highly purified foodingredient from the extract of the Stevia rebaudiana Bertoni plant andits use in various food products and beverages.

DESCRIPTION OF THE RELATED ART

Nowadays sugar alternatives are receiving increasing attention due toawareness of many diseases in conjunction with consumption of high-sugarfoods and beverages. However many artificial sweeteners such as dulcin,sodium cyclamate and saccharin were banned or restricted in somecountries due to concerns on their safety. Therefore non-caloricsweeteners of natural origin are becoming increasingly popular. Thesweet herb Stevia rebaudiana Bertoni, produces a number of diterpeneglycosides which feature high intensity sweetness and sensory propertiessuperior to those of many other high potency sweeteners.

The above-mentioned sweet glycosides, have a common aglycon, steviol,and differ by the number and type of carbohydrate residues at the C13and C19 positions. The leaves of Stevia are able to accumulate up to10-20% (on dry weight basis) steviol glycosides. The major glycosidesfound in Stevia leaves are Rebaudioside A (2-10%), Stevioside (2-10%),and Rebaudioside C (1-2%). Other glycosides such as Rebaudioside B, D,E, and F, Steviolbioside and Rubusoside are found at much lower levels(approx. 0-0.2%).

Two major glycosides—Stevioside and Rebaudioside A, were extensivelystudied and characterized in terms of their suitability as commercialhigh intensity sweeteners. Stability studies in carbonated beveragesconfirmed their heat and pH stability (Chang S. S., Cook, J. M. (1983)Stability studies of stevioside and Rebaudioside A in carbonatedbeverages. J. Agric. Food Chem. 31: 409-412.)

Steviol glycosides differ from each other not only by molecularstructure, but also by their taste properties. Usually stevioside isfound to be 110-270 times sweeter than sucrose, Rebaudioside A between150 and 320 times, and Rebaudioside C between 40-60 times sweeter thansucrose. Dulcoside A is 30 times sweeter than sucrose. Rebaudioside Ahas the least astringent, the least bitter, and the least persistentaftertaste thus possessing the most favorable sensory attributes inmajor steviol glycosides (Tanaka O. (1987) Improvement of taste ofnatural sweetners. Pure Appl. Chem. 69:675-683; Phillips K. C. (1989)Stevia: steps in developing a new sweetener. In: Grenby T. H. ed.Developments in sweeteners, vol. 3. Elsevier Applied Science, London.1-43.)

Methods for the extraction and purification of sweet glycosides from theStevia rebaudiana plant using water or organic solvents are describedin, for example, U.S. Pat. Nos. 4,361,697; 4,082,858; 4,892,938;5,972,120; 5,962,678; 7,838,044 and 7,862,845.

However, even in a highly purified state, steviol glycosides stillpossess undesirable taste attributes such as bitterness, sweetaftertaste, licorice flavor, etc. One of the main obstacles for thesuccessful commercialization of stevia sweeteners are these undesirabletaste attributes. It was shown that these flavor notes become moreprominent as the concentration of steviol glycosides increases (PrakashI., DuBois G. E., Clos J. F., Wilkens K. L., Fosdick L. E. (2008)Development of rebiana, a natural, non-caloric sweetener. Food Chem.Toxicol., 46, S75-S82.)

Some of these undesirable properties can be reduced or eliminated bysubjecting steviol glycosides to the reaction of intermoleculartransglycosylation, when new carbohydrate residues are attached toinitial molecule at C13 and C19 positions. Depending on the number ofcarbohydrate residues in these positions the quality and potency of thecompounds taste will vary.

Pullulanase, isomaltase (Lobov S. V., Jasai R., Ohtani K., Tanaka O.Yamasaki K. (1991) Enzymatic production of sweet stevioside derivatives:transglycosylation by glucosidases. Agric. Biol. Chem. 55: 2959-2965),β-galactosidase (Kitahata S., Ishikawa S., Miyata T., Tanaka O. (1989)Production of rubusoside derivatives by transglycosylation of variousβ-galactosidase. Agric. Biol. Chem. 53: 2923-2928), and dextransaccharase (Yamamoto K., Yoshikawa K., Okada S. (1994) Effectiveproduction of glucosyl-stevioside by α-1,6-transglucosylation of dextrandextranase. Biosci. Biotech. Biochem. 58: 1657-1661) have been used astransglycosylating enzymes, together with pullulan, maltose, lactose,and partially hydrolyzed starch, respectively, as donors of glycosidicresidues.

The transglucosylation of steviol glycosides was also performed byaction of cyclodextrin glucanotransferases (CGTase) produced by Bacillusstearothermophilus (U.S. Pat. Nos. 4,219,571, and 7,807,206) as a resultα-1,4-glucosyl derivatives were formed with degree of polymerization upto 10.

It has to be noted also that many glucosyl stevia products contain up to20% residual dextrins and impurities, such as various polyphenolic,flavonoid compounds and their glycosyl derivatives, etc. Theseadmixtures do not possess significant functional properties, reduce thecontent of steviol glycosides in the product, and some of them affectthe taste profile of final preparation.

The removal of these impurities using polymeric adsorbent resins usuallyyields products with total steviol glycosides' content of up to 90%,which means that ordinary adsorption—desorption technique can achieveonly partial increase of total steviol glycosides' content. Theremaining admixtures still affect the final product sensorycharacteristics limiting their usage in food applications.

Therefore it is necessary to develop highly purified glucosyl steviaproducts with lowest content of admixtures, which will provide the bestcombination of sweetness potency and flavor profile.

SUMMARY OF THE INVENTION

The present invention is aimed to overcome the disadvantages of existingStevia sweeteners. The invention describes a process for producing ahigh purity food ingredient from the extract of the Stevia rebaudianaBertoni plant and use thereof in various food products and beverages asa sweetness and flavor modifier.

The invention, in part, pertains to an ingredient comprisingglucosylated derivatives of steviol glycosides of Stevia rebaudianaBertoni plant. The steviol glycodsides are selected from the groupconsisting of stevioside, Rebaudioside A, Rebaudioside B, RebaudiosideC, Rebaudioside D, Rebaudioside E, Rebaudioside F, dulcoside A,steviolbioside, rubusoside, as well as other steviol glycosides found inStevia rebaudiana Bertoni plant and mixtures thereof.

The invention, in part, pertains to a process for producing aningredient containing glucosylated forms of stevioside, Rebaudioside A,Rebaudioside B, Rebaudioside C, Rebaudioside D, Rebaudioside E,Rebaudioside F, dulcoside A, steviolbioside, rubusoside, as well asother steviol glycosides found in Stevia rebaudiana Bertoni plant. Theprocess can be an enzymatic transglucosylation process using CGTasesproduced by cultures of Bacillus stearothermophilus. The process canalso have a step of post-transglucosylation enzymatic treatment usingvarious glucosidases. The process can also have the steps ofdecolorizing, desalting and removing dextrins and various impurities.The decolorizing can be performed using activated carbon. The desaltingcan be performed by passing through ion exchange resins and/or membranefilters. Removing the dextrins and impurities can be performed bypassing through macroporuos polymeric resin.

In the invention, Stevia extract commercialized by PureCircle (JiangXi)Co., Ltd. (China), containing stevioside (28-30%), Rebaudioside A(50-55%), Rebaudioside C (9-12%), Rebaudioside F (1-3%) and otherglycosides amounting to total steviol glycosides' content of at least95%, was used as a starting material. Alternatively stevia extracts withdifferent ratio of steviol glycosides as well as highly purified steviolglycosides such as Rebaudioside A, stevioside, Rebaudioside D,rubusoside etc, may be used as starting materials.

The starting material was subjected to the enzymatic transglucosylationby action of cyclodextrin glycosyltransferase (CGTase) in the presenceof starch as a glucose donor. As a result α-1,4-glucosyl derivatives ofinitial steviol glycoside were formed. Then the reaction mixture wassubjected to treatment with α-amylase to reduce the number of impuritiescapable of adsorbing on macroporous adsorbent resin in furtherprocessing steps.

The impurities from obtained reaction mixture were removed by AmberliteXAD7 HP resin, and then purified reaction mixture was decolorized,deionized, concentrated and spray dried.

The obtained products were applied in various foods and beverages assweeteners, sweetener enhancers and flavor modifiers, including icecream, cookies, bread, fruit juices, milk products, baked goods andconfectionary products.

It is to be understood that both the foregoing general description andthe following detailed description are exemplary and explanatory and areintended to provide further explanation of the invention as claimed.

BRIEF DESCRIPTION OF THE DRAWINGS

None.

DETAILED DESCRIPTION OF THE INVENTION

Advantages of the present invention will become more apparent from thedetailed description given hereinafter. However, it should be understoodthat the detailed description and specific examples, while indicatingpreferred embodiments of the invention, are given by way of illustrationonly, since various changes and modifications within the spirit andscope of the invention will become apparent to those skilled in the artfrom this detailed description.

Stevia extract commercialized by PureCircle (JiangXi) Co., Ltd. (China),containing stevioside (28-30%), Rebaudioside A (50-55%), Rebaudioside C(9-12%), Rebaudioside F (1-3%) and other glycosides (hereinaftercollectively, “steviol glycosides”) amounting to total steviolglycosides content of at least 95%, was used as a starting material.Alternatively stevia extracts with different ratio of steviol glycosidesas well as highly purified steviol glycosides such as Rebaudioside A,stevioside, Rebaudioside D, rubusoside etc, may be used as startingmaterials.

The HPLC analysis of the raw materials and products was performed onAgilent Technologies 1200 Series (USA) liquid chromarograph, equippedwith Zorbax-NH₂ (4.6×250 mm) column. The mobile phase wasacetonitrile-water gradient from 80:20, v/v (0-2 min) to 50:50, v/v(2-70 min). A diode array detector set at 210 nm was used as thedetector.

The total steviol glycosides content was determined according to“α-Glucosyltransferase Treated Stevia”, Japan's Specifications andStandards for Food Additives, 8th edition, 2009. p 257-258.

The transglucosylation was accomplished by cyclomaltodextringlucanotransferases (CGTases; EC 2.4.1.19) produced by Bacillusstearothermophilus St-88 (PureCircle Sdn Bhd Collection of IndustrialMicroorganisms—Malaysia). However, any other CGTase or enzyme possessingintermolecular transglucosylation activity may be applied as well. Theenzyme can be in a form of cell-free culture broth, concentrated liquidcell-free culture broth, spray dried or freeze dried cell-free culturebroth, or high purity protein. Free and immobilized enzyme preparationscan be used.

The activity of CGTase preparations was determined according to theprocedure described in Hale W. S., Rawlins L. C. (1951) Amylase ofBacillus macerans. Cereal Chem. 28, 49-58.

Starches of different origin may be used as donors of glucosyl unitssuch as, derived from wheat, corn, potato, tapioca, and sago.

Starch was subjected to partial hydrolysis (liquefaction) prior to thetransglycosylation reaction. The dextrose equivalent of the partiallyhydrolyzed starch can be in the range of about 10-25, preferably about12-16. Any enzyme capable of starch hydrolysis may be used forliquefaction, such as α-amylases, β-amylases etc. In one embodiment,CGTase and α-amylase mixtures as liquefying enzymes are preferred.

α-Amylase activity is expressed in Kilo Novo α-amylase Units (KNU). OneKNU is the amount of α-amylase which, under standard conditions (pH 7.1;37° C.), dextrinizes 5.26 g starch dry substance per hour.

The liquefaction mixture contains about 0.001-0.2 KNU, preferably about0.05-0.1 KNU of α-amylase per one unit of CGTase.

The use of α-amylase in liquefaction allows achieving higher throughputsin further activated carbon filtration. When the CGTase is used as theonly liquefying enzyme the filtration rate is approximately 10-15 L/hrper 1 m² of filter surface. In case of liquefaction enzyme mixture(comprising α-amylase and CGTase) the filtration rate is twice asfast—approximately 20-30 L/hr per 1 m² of filter surface.

The ratio of starch and CGTase in the liquefaction mixture is about0.1-0.5 units per one gram of starch, preferably about 0.2-0.4 units pergram.

The concentration of starch in liquefaction mixture is about 15-40%(wt/wt), preferably about 20-30%.

The liquefaction is conducted at about 70-90° C. during about 0.5-5hours, preferably about 1-2 hours.

After liquefaction, the reaction mixture is subjected to thermalinactivation of α-amylase at low pH conditions. The preferred pH rangefor inactivation is about pH 2.5 to pH 3.0 and preferred temperature isabout 95-105° C. The duration of thermal inactivation is about 5-10minutes.

After the inactivation, the pH of the reaction mixture is adjusted toabout pH 5.5-6.5 and the steviol glycosides are added to the mixture anddissolved. The preferred ratio of steviol glycosides to starch (kg ofsteviol glycosides per 1 kg of starch) is about 0.5-1.5, preferablyabout 0.8-1.2.

A second portion of CGTase preparation is added and thetransglucosylation reaction is conducted at about 65° C. for about 24-48hours. The amount of the second portion of CGTase is about 0.2-4 unitsof CGTase per gram of solids, preferably about 0.5-1.2 units per gram ofsolids.

Upon completion of transglucosylation reaction, the reaction was stoppedby heating at about 95° C. for about 15 minutes to inactivate theCGTase, and the solution was treated a second portion of α-amylase ofabout 0.01-0.2 KNU, preferably about 0.05-0.1 KNU per gram of solids wasadded and the reaction was continued for about 12-16 hours at about55-95° C., preferably about 65° C. α-Amylase derived from Bacilluslicheniformis St-3501 (PureCircle Sdn Bhd Collection of IndustrialMicroorganisms—Malaysia) was used in this stage. However α-amylases andother glucosidases derived from any other source may be used as well.

Subsequently the reaction mixture was subjected to thermal inactivationof α-amylase at low pH conditions. The preferred pH range forinactivation is about pH 2.5 to pH 3.0 and preferred temperature isabout 95-105° C. The duration of thermal inactivation is about 5-10minutes. Then the solution was treated with activated carbon, to obtaindecolorized reaction mixture. The amount of activated carbon was about0.02-0.4 grams per gram of solids, preferably about 0.05-0.2 grams pergram of solids.

The decolorized reaction mixture was desalted by passing through ionexchange resins, such as Amberlite FPC23 (H⁺ type) and Amberlite FPA51(OH⁻ type). Other appropriate decolorizing and desalting methods, suchas membrane filtration, or other methods known in the art can be used.

The desalted reaction mixture was further concentrated by vacuumevaporator and dried by means of a spray dryer. Other appropriateconcentrating and drying methods, such as membrane filtration, freezedrying, or other methods known to art can be used. The resulting productcontains non-modified glycosides, α-1,4-glucosyl derivatives andimpurities (Sample 1).

In order to prepare a product with higher content of total sweetglycosides (the sum of glycosylated and non-glycosylated glycosides),the impurities were removed using Amberlite XAD7 HP prior to thedesalting treatment. The steviol glycosides and their glucosylatedderivatives were adsorbed on the resin and subsequently eluted byaqueous ethanol. The resulted aqueous ethanol eluate, containingglucosyl steviol glycosides, was subsequently decolorized and desaltedas described above and the glycosides solution, after the evaporation ofeluting solvent, was powdered by spray drying. The resulting highlypurified glucosyl stevia product contains non-modified glycosides, andα-1,4-glucosyl derivatives (Sample 2).

Using a similar process as for Sample 2, with exclusion of thepost-transglucosylation α-amylase treatment stage, another high purityglucosyl stevia product, containing non-modified glycosides andα-1,4-glucosyl-derivatives, was prepared (Sample 3).

The composition of the samples is summarized in Table 1.

TABLE 1 Composition of glucosyl steviol glycosides samples Content, %Compounds Sample 1 Sample 2 Sample 3 Stevioside 1.7 2.1 1.9 RebaudiosideC 0.9 1.1 1.0 Rebaudioside A 3.9 4.7 4.4 Monoglucosyl-stevioside(StevG1) 8.9 10.8 10.1 Monoglucosyl-Rebaudioside A 11.8 14.3 13.2(RebAG1) Diglucosyl-stevioside (StevG2) 7.6 9.2 8.5Diglucosyl-Rebaudioside A 9.9 12.1 11.1 (RebAG2) Triglucosyl-stevioside(StevG3) 6.1 7.3 6.7 Triglucosyl-Rebaudioside A 7.2 8.5 7.9 (RebAG3)Tetraglucosyl-stevioside (StevG4) 3.3 3.9 3.7 Tetraglucosyl-RebaudiosideA 5.2 6.2 5.8 (RebAG4) Higher glucosylated derivatives 13.9 16.9 15.6Total content of glycosides 80.4 97.1 89.9

The sensory assessment of samples was carried using aqueous solutions,with 20 panelists. Based on overall acceptance the most desirable andmost undesirable samples were chosen. The results are shown in Table 2.

TABLE 2 Sensory assessment of samples in water system Judgment Sample 1Sample 2 Sample 3 Most desirable 2 13 5 Most undesirable 13 0 7Sweetness power 120 160 140 Comments Sweet, Sweet, light, Sweet,slightly soft, round, slightly bitter, pleasant, bitter, astringent,similar to astringent, slight lingering sucrose, no no lingeringaftertaste, lingering aftertaste, sweetness onset aftertaste, sweetnessonset is slow sweetness onset is moderate is rapid

As apparent from the results in Table 2, the sweetness quality of theSample 2 was rated as most superior. Overall the samples with highertotal steviol glycoside content possessed better taste profile.

Sample 2 had the highest sweetness power (160 times sweeter compared toa 5% sucrose solution).

The compositions of this invention can be used as sweetness enhancers,flavor enhancers and sweeteners in various food and beverage products.Non-limiting examples of food and beverage products include carbonatedsoft drinks, ready to drink beverages, energy drinks, isotonic drinks,low-calorie drinks, zero-calorie drinks, sports drinks, teas, fruit andvegetable juices, juice drinks, dairy drinks, yoghurt drinks, alcoholbeverages, powdered beverages, bakery products, cookies, biscuits,baking mixes, cereals, confectioneries, candies, toffees, chewing gum,dairy products, flavored milk, yoghurts, flavored yoghurts, culturedmilk, soy sauce and other soy base products, salad dressings,mayonnaise, vinegar, frozen-desserts, meat products, fish-meat products,bottled and canned foods, tabletop sweeteners, fruits and vegetables.

Additionally the compositions can be used in drug or pharmaceuticalpreparations and cosmetics, including but not limited to toothpaste,mouthwash, cough syrup, chewable tablets, lozenges, vitaminpreparations, and the like.

The compositions can be used “as-is” or in combination with othersweeteners, flavors and food ingredients.

Non-limiting examples of sweeteners include steviol glycosides,stevioside, Rebaudioside A, Rebaudioside B, Rebaudioside C, RebaudiosideD, Rebaudioside E, Rebaudioside F, dulcoside A, steviolbioside,rubusoside, as well as other steviol glycosides found in Steviarebaudiana Bertoni plant and mixtures thereof, stevia extract, Luo HanGuo extract, mogrosides, high-fructose corn syrup, corn syrup, invertsugar, fructooligosaccharides, inulin, inulooligosaccharides, couplingsugar, maltooligosaccharides, maltodextins, corn syrup solids, glucose,maltose, sucrose, lactose, aspartame, saccharin, sucralose, sugaralcohols.

Non-limiting examples of flavors include lemon, orange, fruity, banana,grape, pear, pineapple, bitter almond, cola, cinnamon, sugar, cottoncandy, vanilla flavors.

Non-limiting examples of other food ingredients include flavors,acidulants, organic and amino acids, coloring agents, bulking agents,modified starches, gums, texturizers, preservatives, antioxidants,emulsifiers, stabilisers, thickeners, gelling agents.

The following examples illustrate various embodiments of the invention.It will be understood that the invention is not limited to thematerials, proportions, conditions and procedures set forth in theexamples, which are only illustrative.

EXAMPLE 1

Preparation of CGTase

A strain of Bacillus stearothermophilus St-88 was inoculated in 2,000liters of sterilized culture medium containing 1.0% starch, 0.25% cornextract, 0.5% (NH₄)₂SO₄, and 0.2% CaCO₃ (pH 7.0-7.5) at 56° C. for 24hrs with continuous aeration (2,000 L/min) and agitation (150 rpm). Theobtained culture broth was filtered using Kerasep 0.1 μm ceramicmembrane (Novasep, France) to separate the cells. The cell-free permeatewas further concentrated 2-fold on Persep 10 kDa ultrafilters (Orelis,France). The activity of the enzyme was determined according to Hale,Rawlins (1951). A crude enzyme preparation with activity of about 2unit/mL was obtained.

EXAMPLE 2

Preparation of α-Amylase

A strain of Bacillus licheniformis St-3501 was inoculated in 400 litersof sterilized culture medium containing 1.0% starch, 1.0% corn extract,0.5% NaCl, 0.1% CaCO₃, and 0.002% MnSO₄ (pH 7.2-7.3) at 56° C. for 24hrs with continuous aeration (400 L/min) and agitation (250 rpm). Theobtained culture broth was filtered using Kerasep 0.1 μm ceramicmembrane (Novasep, France) to separate the cells. The cell-free permeatewas further concentrated 100-fold on Persep 10 kDa ultrafilters (Orelis,France). A crude enzyme preparation with activity of about 100 KNU/mLwas obtained.

EXAMPLE 3

Preparation of Glucosyl Stevia Composition

100 g of tapioca starch was suspended in 300 mL of water (pH 6.5). 2 KNUof α-amylase obtained according to EXAMPLE 2 and 30 units of CGTaseobtained according to EXAMPLE 1 were added, and the liquefaction ofstarch was carried out at 80° C. for about one hour to dextroseequivalent about 15. The pH of reaction mixture was adjusted to pH 2.8by hydrochloric acid and the mixture was boiled at 100° C. during 5minutes to inactivate the enzymes. After cooling to 65° C., the pH wasadjusted to pH 6.0 with sodium hydroxide solution. 100 g stevia extractproduced by PureCircle (JiangXi) Co., Ltd. (China), containingstevioside 29.2%, Rebaudioside A 54.3%, Rebaudioside C 9.0%,Rebaudioside F (1.7%) and other glycosides amounting to total steviolglycosides content of about 96.4% was added to liquefied starch andstirred until a homogeneous solution was obtained. 200 units of CGTasewas added to the solution and the mixture was held at a temperature of65° C. for 24 hours under continuous agitation. The obtained reactionmixture was heated at 95° C. for 15 minutes to inactivate the enzyme,and 14 KNU of α-amylase obtained according to EXAMPLE 2 was added. Thereaction was continued for another 12 hours at 65° C. The pH of reactionmixture was adjusted to pH 2.8 by hydrochloric acid and the mixture wasboiled at 100° C. during 5 minutes to inactivate the enzyme. Aftercooling to 65° C., the pH was adjusted to pH 6.0 with sodium hydroxidesolution. 20 grams of activated carbon was added and the mixture washeated to 75° C. and held for 30 minutes. The mixture was filtered andthe filtrate was diluted with water to 5% solids content and passedthrough columns packed with Amberlite FPC23 (H⁺) and Amberlite FPA51(OH⁻) ion exchange resins. The desalted solution was concentrated at 60°C. under vacuum, and dried into a powder form using laboratory spraydryer. 190 grams of product was obtained (Sample 1).

EXAMPLE 4

Preparation of Highly Purified Glucosyl Stevia Composition

100 g of tapioca starch was suspended in 300 mL of water (pH 6.5). 2 KNUof α-amylase obtained according to EXAMPLE 2 and 30 units of CGTaseobtained according to EXAMPLE 1 were added, and the liquefaction ofstarch was carried out at 80° C. for about one hour to dextroseequivalent about 15. The pH of reaction mixture was adjusted to pH 2.8by hydrochloric acid and the mixture was boiled at 100° C. during 5minutes to inactivate the enzymes. After cooling to 65° C., the pH wasadjusted to pH 6.0 with sodium hydroxide solution. 100 g stevia extractproduced by PureCircle (JiangXi) Co., Ltd. (China), containingstevioside 29.2%, Rebaudioside A 54.3%, Rebaudioside C 9.0%,Rebaudioside F (1.7%) and other glycosides amounting to total steviolglycosides content of about 96.4% was added to liquefied starch andstirred until a homogeneous solution was obtained. 200 units of CGTasewas added to the solution and the mixture was held at a temperature of65° C. for 24 hours under continuous agitation. The obtained reactionmixture was heated at 95° C. for 15 minutes to inactivate the enzyme,and 14 KNU of α-amylase obtained according to EXAMPLE 2 was added. Thereaction was continued for another 12 hours at 65° C. The pH of reactionmixture was adjusted to pH 2.8 by hydrochloric acid and the mixture wasboiled at 100° C. during 5 minutes to inactivate the enzyme. Aftercooling to 65° C., the pH was adjusted to pH 6.0 with sodium hydroxidesolution. 20 grams of activated carbon was added and the mixture washeated to 75° C. and held for 30 minutes. The mixture was filtered andthe filtrate was diluted with water to 5% solids content and passedthrough columns each packed with 4000 mL Amberlite XAD 7HP macroporousadsorbent resin. The columns were washed with 5 volumes of water and 2volumes of 20% (v/v) ethanol. The adsorbed glycosides were eluted with50% ethanol. Obtained eluate was passed through columns packed withAmberlite FPC23 (H⁺) and Amberlite FPA51 (OH⁻) ion exchange resins. Theethanol was evaporated and the desalted and decolorized water solutionwas concentrated at 60° C. under vacuum, then dried into a powder formusing laboratory spray dryer. 149 grams of product was obtained (Sample2).

EXAMPLE 5

Preparation of Purified Glucosyl Stevia Composition

100 g of tapioca starch was suspended in 300 mL of water (pH 6.5). 2 KNUof α-amylase obtained according to EXAMPLE 2 and 30 units of CGTaseobtained according to EXAMPLE 1 were added, and the liquefaction ofstarch was carried out at 80° C. for about one hour to dextroseequivalent about 15. The pH of reaction mixture was adjusted to pH 2.8by hydrochloric acid and the mixture was boiled at 100° C. during 5minutes to inactivate the enzymes. After cooling to 65° C., the pH wasadjusted to pH 6.0 with sodium hydroxide solution. 100 g stevia extractproduced by PureCircle (JiangXi) Co., Ltd. (China), containingstevioside 29.2%, Rebaudioside A 54.3%, Rebaudioside C 9.0%,Rebaudioside F (1.7%) and other glycosides amounting to total steviolglycosides content of about 96.4% was added to liquefied starch andstirred until a homogeneous solution was obtained. 200 units of CGTasewas added to the solution and the mixture was held at a temperature of65° C. for 24 hours under continuous agitation. The obtained reactionmixture was heated at 95° C. for 15 minutes to inactivate the enzyme. 20grams of activated carbon was added and the mixture was heated to 75° C.and held for 30 minutes. The mixture was filtered and the filtrate wasdiluted with water to 5% solids content and passed through columns eachpacked with 4000 mL Amberlite XAD 7HP macroporous adsorbent resin. Thecolumns were washed with 5 volumes of water and 2 volumes of 20% (v/v)ethanol. The adsorbed glycosides were eluted with 50% ethanol. Obtainedeluate was passed through columns packed with Amberlite FPC23 (H⁺) andAmberlite FPA51 (OH⁻) ion exchange resins. The ethanol was evaporatedand the desalted and decolorized water solution was concentrated at 60°C. under vacuum, then dried into a powder form using laboratory spraydryer. 166 grams of product was obtained (Sample 3).

EXAMPLE 6

Low-Calorie Orange Juice Drink

Orange concentrate (35%), citric acid (0.35%), ascorbic acid (0.05%),orange red color (0.01%), orange flavor (0.20%), Rebaudioside A (0.003%)and different glucosyl stevia compositions (0.03%) were blended anddissolved completely in water (up to 100%) and pasteurized. Glucosylstevia compositions were represented by Samples 1, 2, and 3, obtainedaccording to EXAMPLES 3, 4, and 5, respectively.

The sensory evaluations of the samples are summarized in Table 3. Thedata show that the best results can be obtained by using the highlypurified glucosyl stevia composition (Sample 2). Particularly the drinksprepared with Sample 2 exhibited a rounded and complete flavor profileand mouthfeel.

TABLE 3 Evaluation of orange juice drink samples Comments Sample FlavorAftertaste Mouthfeel No. 1 Sweet, licorice notes Slight bitterness andNot acceptable aftertaste No. 2 High quality sweetness, Clean, nobitterness Full pleasant taste similar to and no aftertaste sucrose,rounded and balanced flavor No. 3 High quality sweetness, Clean, almostno Somewhat pleasant taste almost bitterness, no acceptable similar toaftertaste sucrose, rounded and balanced flavor

The same method can be used to prepare juices and juice drinks fromother fruits, such as apples, lemons, apricots, cherries, pineapples,mangoes, etc.

EXAMPLE 7

Low-Calorie Carbonated Beverage

A carbonated beverage according to formula presented below was prepared.

Ingredients Quantity, % Sucrose 5.5 Cola flavor 0.340 ortho-Phosphoricacid 0.100 Sodium citrate 0.310 Sodium benzoate 0.018 Citric acid 0.018Rebaudioside A 0.003 Glucosyl stevia composition 0.05 Carbonated waterto 100

The sensory properties were evaluated by 20 panelists. The results aresummarized in Table 4.

TABLE 4 Evaluation of low-calorie carbonated beverage samples Number ofpanelists detected the attribute Taste attribute Sample No. 1 Sample No.2 Sample No. 3 Bitter taste 17 0 5 Astringent taste 16 0 7 Aftertaste 180 4 Comments Quality of sweet taste Bitter aftertaste Clean Clean (16 of20) (20 of 20) (11 of 20) Overall evaluation Satisfactory SatisfactorySatisfactory (1 of 20) (20 of 20) (9 of 20)

The above results show that the beverage prepared using Samples 2possessed the best organoleptic characteristics.

EXAMPLE 8

Diet Cookies

Flour (50.0%), margarine (30.0%) fructose (10.0%), maltitol (8.0%),whole milk (1.0%), salt (0.2%), baking powder (0.15%), vanillin (0.1%)and different glucosyl stevia compositions (0.03%) were kneaded well indough-mixing machine. The obtained dough was molded and baked in oven at200° C. for 15 minutes. Glucosyl stevia compositions were by representedby Samples 1, 2, and 3, obtained according to EXAMPLES 3, 4, and 5,respectively.

The sensory properties were evaluated by 20 panelists. The best resultswere obtained in samples prepared by highly purified glucosyl steviacomposition (Sample 2). The panelists noted rounded and complete flavorprofile and mouthfeel in cookies prepared with Sample 2.

EXAMPLE 9

Yoghurt

Different glucosyl stevia compositions (0.03%) and sucrose (4%) weredissolved in low fat milk. Glucosyl stevia compositions were byrepresented by Samples 1, 2, and 3, obtained according to EXAMPLES 3, 4,and 5, respectively. After pasteurizing at 82° C. for 20 minutes, themilk was cooled to 37° C. A starter culture (3%) was added and themixture was incubated at 37° C. for 6 hours then at 5° C. for 12 hours.

The sensory properties were evaluated by 20 panelists. The best resultswere obtained in samples prepared by highly purified glucosyl steviacomposition (Sample 2). The panelists noted rounded and complete flavorprofile and mouthfeel in sample prepared with Sample 2.

It is to be understood that the foregoing descriptions and specificembodiments shown herein are merely illustrative of the best mode of theinvention and the principles thereof, and that modifications andadditions may be easily made by those skilled in the art withoutdeparting for the spirit and scope of the invention, which is thereforeunderstood to be limited only by the scope of the appended claims.

I claim:
 1. A process for producing a highly purified glucosyl steviacomposition, comprising the steps of: adding starch into water to form astarch suspension; adding a mixture of α-amylase and CGTase into thestarch suspension and incubating for about 0.5 to 2 hours at about75-80° C., resulting in a liquefied starch suspension; inactivating theα-amylase by low pH heat treatment; cooling the liquefied starchsuspension and adjusting the pH to about 5.5 to 7.0; adding steviolglycosides into the liquefied starch suspension, resulting in a reactionmixture; adding a second batch of CGTase into the reaction mixture andincubating for about 12 to 48 hours at about 55-75° C.; adding a secondbatch of α-amylase into the reaction mixture and incubating for afurther about 12-16 hours at about 55-95° C.; inactivating the secondbatch of α-amylase in the reaction mixture by low pH heat treatment; andremoving impurities from the reaction mixture by contacting the reactionmixture with macroporous adsorbent resin and subsequently elutingadsorbed diterpene glycosides with aqueous ethanol to result in apurified reaction mixture; wherein the purified reaction mixturecomprises α-1,4-glucosyl derivatives, and unmodified steviol glycosides.2. The process of claim 1, wherein removing impurities is conducted witha plurality of sequentially connected columns packed with a macroporousadsorbent resin, followed by washing the columns with water, thenwashing with about 10-50% (v/v) ethanol, disconnecting the columns, andeluting each column individually with 30-100% ethanol.
 3. The process ofclaim 1, further comprising decolorizing the purified reaction mixture.4. The process of claim 3, wherein said decolorizing is performed usingion exchange resins or membranes, said membranes being selected from thegroup consisting of ultrafiltration, nanofiltration and reverse osmosismembranes.
 5. The process of claim 1, further comprising desalting thepurified reaction mixture.
 6. The process of claim 5, wherein desaltingis performed by passing the eluate through columns packed with ionexchange resins or membranes, said membranes being selected from thegroup consisting of ultrafiltration, nanofiltration, and reverse osmosismembranes.
 7. The process of claim 1, further comprising concentratingand drying the purified reaction mixture.
 8. The process of claim 1,wherein the weight of the added steviol glycosides is about equal to theweight of the starch.
 9. The process of claim 1, wherein the purifiedreaction mixture comprises at least about 95% total steviol glycosideson an anhydrous basis.
 10. The process of claim 1, further comprisingthe step of adding the highly purified glucosyl stevia composition to asweetening agent to result in a sweetening composition.
 11. The processof claim 1, further comprising the step of adding the highly purifiedglucosyl stevia composition to a flavoring agent to result in a flavorcomposition.
 12. The process of claim 1, further comprising the step ofadding the highly purified glucosyl stevia composition to a foodingredient, selected from the group consisting of acidulants, organicacids, amino acids, coloring agents, bulking agents, modified starches,gums, texturizers, preservatives, antioxidants, emulsifiers,stabilizers, thickeners, gelling agents, and combinations thereof.